Project Summary Metastatic melanomas are aggressive tumors that respond poorly to chemotherapy and radiation and promptly acquire resistance to targeted therapy. While immune checkpoint inhibitors (ICIs) can induce sustained melanoma remission, nearly half of patients are intrinsically resistant to ICIs, in part because their tumors are too large to be cleared by the immune system or are immunologically ?cold? and lack immune recognition necessary to generate anti-tumor T effector cells. Our preliminary data show that senescence- inducing therapy enhances melanoma-cell intrinsic pro-inflammatory program driven by transcription factor NF- ?B. We identified the RIG-I-Like Receptor (RLR) pathway as a candidate NF-?B inducer. This pro-inflammatory program involves enhanced antigen presentation and secretion of chemokines that recruit antigen-presenting cells into the tumor. We also found that senescent cells overexpress checkpoint molecules PD-L1 and Galectin 9, which can inhibit T cells by binding their inhibitory receptors PD-1 and TIM-3, respectively. This suggests that senescence-inducing therapy can enhance recognition of tumor antigens while targeting senescence- specific immune checkpoints can promote adoptive T cell responses against these antigens. Despite its immunogenic properties, therapeutic application of senescence-inducing agents is complicated by the reports that persistent senescent cells may promote tumor metastasis, relapse, and chemo-resistance. Here we hypothesize that we can safely improve melanoma immunogenicity by inducing tumor cell senescence for a limited time, followed by elimination of persistent senescent cells using senolytic drugs that specifically kill senescent cells. This novel therapeutic approach is expected to be effective by: 1) directly blocking tumor cell growth and survival, 2) activating RLR-mediated tumor cell intrinsic immuno-stimulatory signals, and 3) making tumor cells vulnerable to inhibitors of PD-L1/PD1 and Galectin 9/TIM3 immune checkpoints. Three specific aims will examine these mechanisms. In the first aim we will establish the optimal dosing schedule for combined senescence-inducing and senolytic therapy and determine the direct anti-tumor efficacy in an immune-deficient mouse model. A pre-clinical trial using our extensively characterized melanoma PDXs will be conducted to estimate response rate to senescence-inducing and senolytic drug combinations and to identify response-predictive biomarkers. Second aim will investigate the mechanisms whereby senescence-inducing and senolytic therapy facilitates the innate immune sensing of tumor focusing on the role of RLRs. We will examine whether deficiency in RLR signaling can abrogate NF-?B activation, chemokine secretion, and immune cell recruitment into senescent tumors. Aim three will determine if senescence-inducing and senolytic therapy augments immune checkpoint inhibitor (ICI) responses. We will utilize anti-PD-L1 and anti-TIM3 agents to target senescence-cell specific immune checkpoints. These studies will provide a preclinical basis for clinical development of rational combinations of senescence-inducing and senolytic agents.